A fluorescence microplate assay using yopro-1 to measure apoptosis: application to HL60 cells subjected to oxidative stress.

A new one-step labeling procedure using the membrane permeant fluorescent probe yopro-1 in association with fluorescence microtitration for the rapid determination of apoptosis is reported. Programmed cell death was induced by the pro-apoptotic agents etoposide and staurosporine, and measured in nonadherent HL60 cells and adherent phorbol 12-myristate 13-acetate (PMA)-treated HL60 cells. Cell viability was controlled by trypan blue exclusion and calcein-AM staining. To confirm results of fluorescence microplate assay, apoptosis was measured by flow cytometry analysis using the same fluorescent probe, and results showed corresponding data between both procedures. Development of apoptosis was confirmed by the presence of PARP (poly(ADP-ribose) polymerase cleavage and nuclear DAPI (4,6-diamidino-2-phenylindole) staining, two well-known methods used to investigate apoptosis. The fluorescence microplate assay was also applied to measure apoptosis in cells exposed to an oxidative stress induced by tert-butylhydroperoxide (t-BHP), and results confirmed the potential of the fluorescence microplate assay in measuring events of apoptosis, especially in adherent, cultured, living cells.

Activity of proximal promoter of the human beta(1)-integrin gene was increased in Sézary syndrome.

Changes in beta1-integrin expression have been involved in abnormal cellular interactions between malignant lymphocytes from Sézary (Sz) patients and keratinocytes. In this paper, we compare the activity of both distal and proximal promoters of the beta1-integrin gene in malignant lymphocytes from Sz patients with human normal lymphocytes. Activity of both beta1-integrin promoters was also analysed in human normal keratinocytes. Northern blot analysis shows that beta1-integrin mRNA expression is higher in malignant Sz lymphocytes than in normal lymphocytes. CAT assays show that the activity of proximal beta1-integrin promoter is markedly increased (up to 6-fold) in malignant lymphocytes from Sz patients, in comparison to normal lymphocytes. These results suggest that changes in activity of the proximal promoter of beta1-integrin subunit could be, in part, responsible for the abnormal cellular interactions between malignant lymphocytes and keratinocytes observed in Sz syndrome.

Phorbol ester treatment of HL 60 leukemia cells results in increase of beta-(1 --> 4)-galactosyltransferase.

We previously showed that HL 60 leukemia cells exhibit various changes in their cellular glycans after phorbol 12-myristate 13-acetate (PMA) treatment. These changes could originate largely from changes in one or several glycosyltransferases. In this report, we show using enzymatic measures, fluorescence microscopy, immunoblotting and Northern blot that beta-(1 --> 4)-galactosyltransferase I (GalT I) activity was higher (> x 2) in PMA-treated compared with untreated HL 60 cells. Immunoblotting showed an increased intensity of the GalT I band at 49 kDa and Northern blot a weak increase of the GalT I transcript band, after PMA treatment. Moreover, Northern blot performed after actinomycin-D treatment of the cells, which inhibits transcription, suggests that the observed increase of GalT I expression could originate, in part, from increase of the stability of GalT I transcripts.

Use of fluorescent probes to assess the early sulfhydryl depletion and oxidative stress induced by mechlorethamine in human bronchial epithelial cells.

In this study, we report in vitro methods using fluorescent probes to assess thiol depletion and the oxidative stress induced by mechlorethamine (HN2), a nitrogen mustard, on a human bronchial epithelial cell line (16HBE14o-). Monobromobimane (mBBr) and 2',7'-dichlorofluorescin-diacetate (H(2)DCF-DA) were respectively used to monitor the intracellular thiol and peroxide levels. Fluorescent measurements were realized on gated live cells with a flow cytometer and a microspectrofluorimeter. Results clearly show that HN2 induced an early reduction of free sulfhydryl groups inside the cell correlated with an increase in the intracellular peroxides content. HN2 effects were time and dose dependent. The use of these fluorescent probes provide a useful and rapid methods for future screening of protective molecules against the early sulfydryl depletion and oxidative stress induced by HN2 on human airway epithelium.

T lymphocytes from Sézary syndrome patients express beta1 integrins whose beta(1-6)-branched N-linked oligosaccharides reflect their adhesive capacity.

Sézary syndrome (Sz), characterized by slowly progressing clonal proliferation of CD4+, CD45 RO+ T cells, has several forms that are distinguished according to the epidermotropic properties of the pathological cells. In a recent paper (Derappe C, Haentjens G, Lemaire S, Feugeas JP, Lebbe C, Pasqualetto V, Bussel A, Aubery M, Néel D. Leukemia 1996;10:138), we observed that T lymphocytes from most of the Sézary patients [Szbeta(1-6)+] expressed high levels of beta(1-6)-GlcNAc-branched N-linked oligosaccharides while T lymphocytes from other patients [Szbeta(1-6)-] did not. Because this observation suggests the possibility of two forms of Sz, distinguished according to the expression rate of these glycans, we looked for a possible relationship between this expression rate and T-cell adhesiveness. Using an original protocol (Braut-Boucher F, Pichon J, Rat P, Adolphe M, Aubery M, Font J. J Immunol Methods 1995;178:41), we observed that T lymphocytes obtained from the Szbeta(1-6)+ patients adhered less to normal keratinocyte monolayers than T lymphocytes from Szbeta(1-6)- patients and normal donors. As assessed by FACS analysis, all the integrin-subunits studied were more expressed on Szbeta(1-6)-, especially alpha4, alpha5, beta1 and beta2, than on Szbeta(1-6)+ and normal lymphocytes. Although these results suggest that beta1- and beta2-integrin expression is involved in the adhesive properties of these T-cells, other factors, such as glycosylation, may also contribute. To demonstrate this possibility, we sought the presence of beta(1-6)-GlcNAc-branched N-linked oligosaccharides on beta1 integrins expressed by T lymphocytes from Sz patients. Immunoblot experiments, performed using the specific lectin from Phaseolus vulgaris (Leukoagglutinin form), showed that only the beta1 integrin subunit expressed by T lymphocytes from Szbeta(1-6)+ patients carried these glycans, supporting the concept of the involvement of T-cell glycosylation in the evolution of Sz.

Integrins of the beta1 family influence keratinocyte-lymphocyte interaction.

Data from the literature indicate that ICAM-1 molecules play an important role in keratinocyte interactions with lymphocytes via the lymphocyte function-associated-1 lymphocyte-adhesion molecule. We examined the role of beta1 integrins in keratinocyte-lymphocyte adhesion under different activation conditions. Among the beta1 integrins expressed on keratinocytes and lymphocytes detected by indirect immunofluorescence microscopy and flow cytofluorometry, primarily the alpha2 and the alpha3 subunits on both cell types were involved in keratinocyte-lymphocyte adhesion. Moreover, the highest adhesion level was observed when both cell types were activated by IFN-gamma for keratinocytes and phorbol 12-myristate 13-acetate for lymphocytes, suggesting that the former involved the protein kinase C pathway. Keratinocyte activation, characterized by the expression of ICAM-1, a decrease of beta1 integrins, and the absence of alpha5beta1 integrin, was required for optimal lymphocyte adhesion. Thus, beta1 integrins remaining at the surface of IFN-gamma-treated keratinocytes could be activated by this cytokine, and could synergize with ICAM-1 and lymphocyte function-associated-1 molecules to consolidate keratinocyte-lymphocyte adhesion.

Atypical microtubule organization in undifferentiated human colon cancer cells.

We previously reported that undifferentiated colonic cancer HT-29 cells, unlike the differentiated ones, exhibit unusual organelle distributions and atypical vesicle trafficking patterns, which are microtubule-independent and microfilament-dependent. In the present study, we have analyzed the microtubule network in both phenotypes, using confocal microscopy, and determined the expression levels of some microtubule-associated proteins by quantitative immunoblotting. Differentiated cells exhibited the microtubular organization of polarized epithelial cells. Non-polarized undifferentiated cells presented an atypical microtubule organization as microtubules were localized mainly at the cell 'top'. Immunoblot analysis indicated the absence or low content of several structural and motor microtubule-associated proteins in undifferentiated cells, compared to differentiated cells. This may explain in part their atypical microtubular organization. This study agrees with a crucial role for microfilaments in the intracellular organization of undifferentiated HT-29 cancer cells, while differentiated HT-29 cells exhibit intracellular organization similar to that of normal enterocytic cells, although they are also tumoral.

T lymphocyte activation results in an increased expression of beta-1,4-galactosyltransferase: phorbol ester induces a similar enhancement in the absence of mitosis.

We previously showed that in vitro activated human T lymphocytes expressed increased amounts of beta-1,6-branched N-linked oligosaccharides (Lemaire S etal. (1994) J Biol Chem269: 8069-74), which have been proposed to participate in the regulation of the immune process. In the present paper, we compared the activity and expression of beta-1,4-galactosyltransferase (GalT), one of the glycosyltransferases involved in the biosynthesis of these beta-1,6-branched N-linked oligosaccharides, before and after in vitro activation of T lymphocytes after a 40h treatment with a mixture of phorbol 12-myristate 13-acetate and Phaseolus vulgaris lectin. After treatment, the enzymatic activity of the GalT was significantly increased and immunoblot experiments performed with a monoclonal antibody to human GalT showed an increased intensity of the GalT band at 49 kDa, attributable to an enhancement of GalT mRNA level, as shown by Northern blots. However, treatment of the same T-lymphocytes by phorbol ester alone, which is unable to induce mitosis, resulted in a comparable increase of the expression of GalT. Moreover, these phorbol ester-treated T lymphocytes, analysed by flow cytometry exhibited a two-fold increase in the expression of GalT. Finally, confocal fluorescence microscopy performed on all T lymphocytes (treated or not) showed that the flow cytometric signal of GalT originates from intracellular, Golgi-associated antigen only since no surface GalT was detected.

Altered collagen of human pathological fibroblasts impairs the synthesis of fibronectin.

Human fibroblasts with mutated type I collagen have marked defective adhesive capacities on exogenous type I collagen and exogenous fibronectin in comparison to normal fibroblasts. This defective cell adhesion could be partly explained by the decreased level of cell surface receptors of the beta 1-integrin family, i.e., the alpha 2 integrin subunit for type I collagen and the alpha 5 integrin subunit for fibronectin, observed in pathological fibroblasts. However, it appeared that the presence of altered collagen interfered both with fibronectin biosynthesis and with its surface expression. Using a binding assay on immobilized fibronectin, we demonstrated that the mutated collagen had a weaker binding to fibronectin. In addition, the pathological fibroblasts plated on a mixture of normal exogenous type I collagen and fibronectin exhibited the same maximal level of adhesion as control fibroblasts. These results indicate that fibroblasts with the mutated collagen exhibit a decreased binding to normal fibronectin, a modification of synthesis and surface expression of fibronectin, and, finally, altered adhesive capacities.

Barrier function of reconstructed epidermis at the air-liquid interface: influence of dermal cells and extracellular components.

To evaluate the epidermal barrier function of in vitro reconstructed epidermis, we measured the penetration of estradiol and water across human keratinocytes cultured in defined medium (DM), in the presence of proliferative fibroblasts (pF) or conditioned medium derived from pF, at the air-liquid interface on synthetic porous membrane, noncoated or coated with laminin, fibronectin, type I collagen or type IV collagen. Ultrastructural analysis showed a well-developed stratum corneum whatever the culture conditions. The permeability of reconstructed epidermis in DM on a noncoated porous membrane was 5- to 10-fold higher than human native epidermis, with both tracers. No significant change in barrier function was observed whatever the culture conditions.

Human keratinocyte models: Assessment of cell adhesion and dermotoxicity using fluorescent probes.

To assess the molecular and cellular events that occur in the skin during biological and pharmaco-toxicological processes, we developed different in vitro models. Two major systems are described: (1) relatively simple ones such as normal human keratinocytes (NHK) grown in monolayer or continuous culture of spontaneously immortalized keratinocyte cells, the HaCaT cell line. This cell line forms a monolayer and displays the same phenotypic morphology, pattern of differentiation markers as NHK. (2) More complex models such as NHK multilayers differentiated on a synthetic porous membrane. Indeed, NHK grown at the air-liquid interface of culture inserts may undergo epidermal differentiation in 21 days (Noël-Hudson et al., 1995a). Under the same culture conditions, no stratification of the HaCaT cell line was obtained. NHK and/or HaCaT monolayers were used to study the cell surface molecules involved in heterologous cell interactions, and to estimate the cytotoxic effects of different compounds through a sensitive fluorimetric microtitration assay. When cell adhesion was measured with calcein-AM labelled lymphocytes, it appeared that lymphocytes display the same behaviour towards NHK or HaCaT cells. The importance of the activation status of each cell and the involvement of alpha(2) and alpha(3)beta(1) integrins in lymphocyte-keratinocyte interactions were demonstrated. Likewise cytotoxicity of SDS and DNP was easily and rapidly detected with calcein-AM and Alamar blue probes. Skin models in combination with fluorescent probes offer promising alternatives for assessing cell interactions as well as cytotoxic effects.

Comparison of six different methods to assess UVA cytotoxicity on reconstructed epidermis. Relevance of a fluorimetric assay (the calcein-AM) to evaluate the photoprotective effects of alpha-tocopherol.

A three-dimensional culture of human keratinocytes exposed at the air-liquid interface has been developed and used in conjunction with fluorimetric, colorimetric and radioligand incorporation assays to assess the in vitro toxicity of UVA. The aims of the study were: (1) to compare the relevance of the neutral red uptake (NR), MTT metabolism, (35)S-methionine incorporation, IL1-alpha release and calcein-AM esterification assays for the evaluation of UVA injury; (2) to test the preventive protective effect of an emulsion containing 3% of tocopherol applied on the reconstructed epidermis, in comparison with an application of tocopherol 3% diluted in culture medium either on the apical compartment or in the underneath compartment of the skin culture insert. Viability measurement methods are based on different endpoints. None of the five endpoints measured produced LD(50) values (40 J/cm(2)) that differed significantly from the others. However, calcein-AM assay was relatively more reproducible and easier to handle than the others, and seemed to be a better choice for the evaluation of the protective effects of the tocopherol emulsion. Tocopherol diluted in culture medium under the epidermis 24 hr before irradiation failed to protect the epidermis against UVA damage, whereas diluted in culture medium or in oily emulsion and applied to the epidermis reduced cellular death (cellular recovery values are, respectively, 24% and 21%). Since cosmetic or pharmaceutical formulations can be directly applied on the reconstructed epidermis as in vivo, this model in combination with a fluorescent viability assay appears to be a suitable approach for pharmaco-toxicological evaluations.

Lectin activities of cytokines and growth factors: function and implications for pathology.

The discovery that certain cytokines have carbohydrate-binding (lectin) properties opens new concepts in the understanding of their mechanism of action. The carbohydrate-recognition domain, which is localized opposite to the receptor-binding domain, makes these molecules bi-functional. The expression of the biological activity of the cytokine relies on its carbohydrate-binding activity which allows the association of the cytokine receptor with molecular complexes comprising the specific kinase involved in receptor phosphorylation and in specific signal transduction. It is expected that blood accumulation of free or membrane-bound glucan ligands of cytokines may dramatically perturb their endogenous function inducing specific immunodeficiencies.

Nuclear and cytoplasmic expressions of the carbohydrate-binding protein CBP70 in tumoral or healthy cells of the macrophagic lineage.

The expression of the carbohydrate-binding protein CBP70 was analyzed in undifferentiated HL60 cells, HL60 cells differentiated into monocytes/macrophages or granulocytes, healthy monocytes and in vitro monocyte-derived macrophages (MDM) using an anti-CBP70 serum. This study was performed by immunoblotting analysis of nuclear and cytoplasmic extracts before and after N-acetylglucosamine affinity chromatography and by indirect immunofluorescence microscopy. The results of this study show, for the first time, that CBP70 is expressed in the nucleus and the cytoplasm of healthy or leukemic cells of the macrophagic lineage. However, striking differences were observed depending upon the leukemic or normal state of cells and cell differentiation. Indeed, the level of expression and the intracellular distribution of CBP70 were found to be different in undifferentiated HL60 cells and monocytes/macrophages differentiated from these cells. Major differences were also observed according to whether macrophages differentiated from leukemic HL60 cells or healthy monocytes. Thus, the total cellular expression of CBP70 was markedly lower in MDM than in HL60-derived macrophages and the intracellular distribution of the protein was different. Nevertheless, in both cases, the total cellular expression of CBP70 was enhanced during cell differentiation. Another important result is the finding that CBP70 behaviour was totally different when HL60 cells were induced to differentiate into macrophages or granulocytes. These data could therefore suggest that CBP70 is involved in phagocytic cell differentiation. Moreover, we show that an additional 60 kDa polypeptide (p60), recognized by the anti-CBP70 serum, is expressed in HL60 cells differentiated into macrophages or granulocytes as well as in healthy monocytes or MDM but not expressed in undifferentiated HL60 cells. Although CBP70 and p60 appeared to be closely related polypeptides, their relationship remains to be precised. These findings are discussed with regard to data available on galectin-3.

Organization of the endoplasmic reticulum-Golgi system is related to the state of enterocytic differentiation of human HT-29 cells.

The organization of the endoplasmic reticulum (ER)-intermediate compartment (IC)-Golgi system was studied in tumoral HT-29 cells. Depending on the culture conditions, these cells are either undifferentiated or exhibit enterocytic differentiation after reaching confluence. In differentiated HT-29 cells, these organelles were organized as in most cell types. They displayed a classical structure and appeared associated with microtubules, as nocodazole altered both their structure and intracellular localization. Likewise, membrane dynamics of the Golgi appeared normal: as in many other cells, brefeldin A (BFA) induced retrograde transport from the Golgi to the ER, demonstrated by tubulation of the Golgi elements and shift of the galactosyltransferase activity from the Golgi- to the RER-enriched fraction, isolated by subcellular fractionation. In contrast, atypical features were observed in undifferentiated HT-29 cells: the Golgi structure exhibited abnormal swellings; the IC elements were very rare. Only cytochalasin D altered the structure and intracellular localization of the three organelles, suggesting that they were associated with microfilaments instead of microtubules. The membrane dynamics were unusual: brefeldin A led to a vesiculation of the Golgi elements with a slowed-down retrograde transport of galactosyltransferase. HT-29 cells engaged in the differentiation process, but which were still undifferentiated, showed mainly the features of undifferentiated cells, with a few characteristics of differentiated cells. These results indicate that the structure of the Golgi apparatus, IC and ER, their relationships to cytoskeletal elements and membrane dynamics depend on the state of differentiation of HT-29 cells. Although they are tumoral, differentiated HT-29 cells exhibit features observed in non-tumoral polarized epithelial cells. On the contrary, undifferentiated HT-29 cells display important abnormalities that may be related to their metastatic properties.

Circulating malignant lymphocytes from Sezary syndrome express high level of glycoproteins carrying beta (1-6)N-acetylglucosamine-branched N-linked oligosaccharides.

The circulating forms of malignant cells from patients with Sezary syndrome exhibit on their glycoproteins a high level of beta (1-6)GlcNAc-branched N-linked oligosaccharides, a particular species of glycans related to the metastatic potential of several tumors and T lymphocytes activation. An increased activity of the N-acetylglucosaminyltransferase V and of the beta (1-4)galactosyltransferase, two enzymes implicated in beta (1-6)GlcNAc-branching is also found. Nevertheless, contrary to activated normal T lymphocytes, Sezary lymphocytes in agreement with their non-proliferating state, do not exhibit increased thymidine uptake. This result suggests that expression of the beta (1-6)GlcNAc-branched N-linked carbohydrates could be related to some of the malignant properties of Sezary lymphocytes.

Ricin toxicity and intracellular routing in tumoral HT-29 cells. I. Ricin routing and toxicity are related to the state of differentiation of HT-29 cells.

We previously showed that ricin, which is more cytotoxic to undifferentiated than to differentiated tumoral HT-29 cells, enters these cells by different routes. The final steps of ricin endocytosis were investigated in order to identify the translocation site from which ricin exerts its toxicity. Toxicity measurements and kinetic experiments followed by subcellular fractionation were run in parallel. In differentiated cells, from 20 min of internalization, radiolabeled ricin was found in a Golgi-enriched fraction. At 60 min, which corresponds to the lag time for ricin toxicity, the amount of radioactivity located in this fraction decreased without any concomitant increase in the other fractions. In undifferentiated cells, from 20 min of incubation, radiolabeled ricin was detected in the ER-enriched fractions. At 30 min, the lag time for ricin toxicity, the amount of radioactivity detected in these fractions decreased without any concomitant increase in the Golgi-enriched fraction. Monensin, which was used to confirm the passage of ricin through the Golgi, greatly increased ricin toxicity and diminished the lag time only in differentiated cells. Brefeldin A inhibited ricin toxicity when added before the end of the lag time in both cell populations and reduced the amount of ricin detected, respectively, in the Golgi- and ER-enriched fractions in differentiated and undifferentiated cells. We propose that ricin enters the cytosol from the Golgi apparatus and essentially from the ER in differentiated and undifferentiated HT-29 cells, respectively, and that these different intracellular routings might explain the differential toxicity of ricin.

Ricin toxicity and intracellular routing in tumoral HT-29 cells. II. Differential ricin toxicity from the apical and basolateral surfaces of differentiated HT-29 cells.

We previously showed that ricin, a toxin commonly used in the construction of immunotoxins, was more toxic to undifferentiated than to differentiated HT-29 tumoral cells. This results from differences in the intracellular routings of the toxin. As these studies concerned the entry through the apical pole of differentiated polarized HT-29 cells, we investigated and compared the intracellular routing of ricin from the apical and basolateral membranes of differentiated HT-29 cells and the toxicity of ricin depending on the pole of administration. For this purpose, we developed the culture of polarized HT-29 cells on porous membrane filters and demonstrated that differentiated HT-29 cells can establish a leakproof monolayer. Ricin is 2.5-fold less toxic when it is added at the basolateral than at the apical pole of the cells, which may result from different observations: (1) less ricin is bound at the basolateral membrane than at the apical one, leading to a lesser internalization of the toxin; (2) ricin sorting in the apical and basolateral endocytic compartments of HT-29 cells differs: apically internalized ricin is targeted intracellularly while basolaterally internalized ricin uses mainly the transcytotic pathway; using NH4Cl and monensin, we observed that ricin follows the same pathway from both sides of the cells, namely the endosomal system, to reach the Golgi apparatus from which toxin intoxication occurs; (3) kinetics studies showed that a delay exists before an efficient intoxication by the basolateral pole is observed. The use of monensin at low concentration in order to perturb only the Golgi functions indicated that this delay could account for a different presentation of the toxin toward the membrane of the apical and basolateral endocytic compartments. Together, our results showed that, in differentiated HT-29 cells, if the pathways carrying ricin from the apical and basolateral membranes to the Golgi apparatus appear identical, ricin exerts differentially its toxicity depending upon the surface of administration, i.e., the apical or the basolateral surface of the cells.

Human lymphocyte activation is associated with the early and high-level expression of the endogenous lectin CSL at the cell surface.

Lymphocytes undergo activation in response to antigens, cytokines, lectins and antibodies interacting with specific cell-surface molecules or through substances influencing signal transduction pathways. This study shows that human T- and B-cells stimulated using phorbol esters or plant lectins express early (2 h using phorbol esters and 24 h using plant lectins) a high level of a polyvalent carbohydrate-binding protein, the cerebellar soluble lectin (CSL), which is in part externalized. The lectin, immunologically related to CDw70, interacts with specific glycoprotein ligands of the lymphocyte surface, including CD3 on T-cells and CD24 on B-cells. Major changes in phosphorylations associated with activation appear as largely CSL-dependent since they are specifically inhibited by anti-CSL Fab fragments. It is suggested that the lectin induces the clustering of specific cell-surface glycoproteins and plays the role of an endogenous amplifier of activation signals.